Research Highlights

When it comes to drug development, membrane proteins play a crucial role, with about 50% of drugs targeting these molecules. Understanding the function of these membrane proteins, which connect to the membranes of cells, is important for designing the next line of powerful drugs. To do this, scientists study model proteins, such as bacteriorhodopsin (bR), which, when triggered by light, pump protons across the membrane of cells. 

While bR has been studied for half a century, physicists have recently developed techniques to observe its folding mechanisms and energetics in the native environment of the cell’s lipid bilayer membrane. In a new study published by Proceedings of the National Academy of Sciences (PNAS), JILA and NIST Fellow Thomas Perkins and his team advanced these methods by combining atomic force microscopy (AFM), a conventional nanoscience measurement tool, with precisely timed light triggers to study the functionality of the protein function in real-time. 

In a new paper, JILA physicist Thomas Perkins collaborated with CU Biochemistry Prof. Marcello Sousa to dissect the mechanisms of how certain bacteria become more virulent. The research brings together the Perkins lab expertise in single-molecule studies and the Sousa lab expertise in the type III secretion system, a key component of Salmonella bacteria. 

"Unraveling" cell membrane proteins could help us understand how to build better drugs and treatments for disease.

It's tough to get tightly-wound balls of DNA to lay down flat and straighten out to get their picture taken. A new technique from the Perkins group gets a crisp, clear picture in just five minutes.

JILA researchers have demonstrated a much easier, faster and more precise way to understand the structure and function of the HIV RNA molecule, especially the HIV RNA hairpin. Furthermore, the techniques developed for this research promise to allow a wider range of users to study similar biological molecules, as they are built upon commercially available and user-friendly atomic force microscopes, or AFMs.

The Perkins group has made dramatic advances in the use of Atomic Force Microscopes (AFMs) to study large single biomolecules, such as proteins and nucleic acids (DNA, RNA), that are important for life. After previously improving AFM measurements of biomolecules by orders of magnitude for stability, sensitivity and time response, the Perkins group has now developed ways to make these precision biomechanical measurements up to 100 times faster than previously possible––obtaining useful information in hours to days rather than weeks to months. 

The Perkins group continues to extend the performance of its unique Atomic Force Microscope (AFM) technology, revealing for the first time a dozen new short-lived intermediate states in the folding and unfolding of a membrane protein that controls the exchange of chemicals and ions into and out of living cells. Measuring the energetics and dynamics of membrane proteins is crucial to understanding normal physiology and disease, and the Perkins group’s observation of multiple new folding/unfolding states shines new light on these cellular “gatekeepers.”

The Perkins Group has demonstrated a 50-to-100 times improvement in the time resolution for studying the details of protein folding and unfolding on a commercial Atomic Force Microscope (AFM). This enhanced real time probing of protein folding is revealing details in these complex processes never seen before. This substantial enhancement in AFM force spectroscopy may one day have powerful clinical applications, including in the development of drugs to treat disease caused by misfolded proteins. Misfolded proteins are implicated in such fatal maladies as Creutzfeldt–Jakob disease and mad cow disease, both of which are caused by prions.

Fellow Tom Perkins’ group is significantly closer to realizing its long-standing dream of using atomic force microscopy (AFM) to study how membrane proteins fold and unfold. Historically, scientists have used AFM to measure the mechanical forces needed to unfold individual proteins and the resulting increase in their lengths. However, the limitations of AFM itself have prevented researchers from watching the unfolding process in detail.

The groups of Fellow Adjoint Markus Raschke and Fellow Tom Perkins joined forces recently to shine light onto a bacterial membrane protein called bacteriorhodopsin (bR). They used a new infrared (IR) light imaging system with a spatial resolution and chemical sensitivity of just a few bR molecules. In their experiment, the tip of an atomic force microscope (AFM) acted like an antenna for the IR light, focusing it onto the sample.

Gold glitters because it is highly reflective, a quality once considered important for precision measurements made with gold-coated probes in atomic force microscopy (AFM). In reality, the usual gold coating on AFM probes is a major cause of force instability and measurement imprecision, according to research done by the Perkins group.

In science, it can be fun and interesting to upend conventional wisdom. A good example is what just happened to a widely accepted explanation for overstretching of double-stranded DNA (dsDNA). Overstretching occurs suddenly when researchers add a tiny increment of force to dsDNA that is already experiencing a pulling force of approximately 65 pN. (A piconewton is a trillionth of a newton, which is roughly equal to the gravitational force on a medium-sized apple). The small additional force causes the dsDNA to suddenly become 70% longer — as it stretches like a slinky.

Atomic force microscopy (AFM) just got a whole lot more efficient for studying proteins and other biomolecules. Graduate student Allison Churnside, former research associate Gavin King, and Fellow Tom Perkins recently used a laser to detect the position of sparsely distributed biomolecules on a glass cover slip. Since the same laser is also used to locate the AFM tip, it is now possible to align the microscope tip and sample with a precision of 40 nm, before the AFM tip even touches the sample. The researchers say that the new sample detection scheme solves the “needle in a haystack” problem of nanoscale microscopy.

The most important step for a microscope wanting to marry another microscope is finding the right partner. A professional matchmaker, such as the Perkins lab, might be just the ticket. The group recently presided over the nuptials of atomic force microscopy and optical-trapping microscopy. Research associate Gavin King, graduate students Ashley Carter and Allison Churnside, CU freshman Louisa Eberle, and Fellow Tom Perkins officiated. The marriage produced an ultrastable atomic force microscope (AFM) capable of precisely studying proteins in real-world (ambient) conditions.

The Perkins group is helping to develop DNA as a force standard for the nano world. Polymers of DNA act like springs, and DNA's elasticity may one day provide a force standard from 0.1–10 piconewtons (pN). One pN is the force exerted when 1 mW of light reflects off a mirror or the approximate weight of one hundred E. coli cells. DNA is an excellent candidate for a force standard because its double helix is reproduced with exquisite fidelity, which allows researchers (or cells) to build it with atomic precision.

Life can be challenging on the biophysics research frontier. Consider gold nanoparticles as a research tool, for example. Gold is ductile and malleable as well as being a good conductor of heat and electricity. Its unique chemistry allows proteins and DNA to be easily attached to these nanoparticles. Physicists have been investigating gold nanoparticles in optical-trapping experiments because they enhance trapping efficiency and potentially increase detection sensitivity.

Lora Nugent-Glandorf and Tom Perkins have come up with an optical trap motion detector that can "see" protein motors moving one base at a time along a DNA helix. For some time scientists have been able to make optical traps that can track the movement of attached beads, but the method had a resolution of 1-2 nanometers, which was not sensitive enough to resolve .338 nm DNA base steps. The lack of resolution was mostly due to instrument drift.